Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan.

This study identified nontuberculous mycobacteria (NTM) recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB) using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32) isolates, 550 bp. (2) isolates and 600 bp. (14) isolates. Four isolates were contaminated.


Introduction
The genus Mycobacterium is represented by a wide range of organisms found both in clinical or environmental material, with complex phenotypic and genetic data, and associated with various human and animal diseases (Shinnick and Good, 1994;Wallace, 1994). Nontuberculous mycobacteria (NTM) have emerged as public health hazards in developing countries where lack of nutrition, civil wars and environmental crisis occur (Mokaddas and Ahmad, 2007). In tropical climates NTM are frequently isolated from diverse environmental sources, many animals and human conditions (Kim et al., 2005). For example, M. farcinogenes and M. sengelenses have been reported causing a chronic infection in zebu cattle namely bovine farcy (BF) (Lechevalier et al., 1971;El Sanousi et al., 1977). BF is an endemic non treatable disease in Africa causes lymphatic and pulmonary suppurative forms resembled to tuberculosis lesions and lead to carcasses condemnations (Chamoiseau, 1979;Mohan, 1985). The prevalence rate of the disease in the Sudan was found as 15% in western Sudan (El Nasri, 1961), 5.19% in Nyala, 4.19% in Nuba Mountains and 3.49% in Omdurman area (El Sanousi et al., 1977, 1979), while EL Tigani-Asil et al. (2013 reported the prevalence rate of BF as 2.2% in South Darfur State. Human infections include M. farcinogenes in hip joint arthroblasty (Wong et al., 2005) and M. Sengelenses in catheter-related bloodstream infection in a Korean cancer patient (Oh et al., 2005) were reported. M. avium was isolated from a Friesian cross breed suffering from parotid and maxillary nodular infections similar to BF lesions, which made a clinical diagnosis difficult (Hamid et al., 2007). Phenotypic identification of mycobacteria grown in culture utilizes biochemical features (Kent and Kubica, 1985) is time-consuming and does not identify closely related taxa (Springer et al., 1996). 16S-23S ribosomal DNA internal transcribed spacers (16S-23S rDNA ITS) have been considered an alternative method for identification (Roth et al., 2000). PCR amplification with primers Ec16S.1390p and Mb23S.44n resulted in the detection of a single band of approximately 480 bp in all 60 Mycobacterium http: //www.openveterinaryjournal.com A.E. El Tigani-Asil et al. Open Veterinary Journal, (2014), Vol. 4(1): 16-19 ________________________________________________________________________________________________________ strains investigated. The variation in product length was not considerable between the different species of slow growers, but a smaller product of approximately 430 bp was noted for M. xenopi strain (Roth et al., 1998). This study aimed to identify NTM from bovine suppurative tissues by the genus specific 16S-23S rDNA ITS gene.

Test strains
Fifty two isolates were included in this study and they were phenotypically identified as farcy agents out of 59 confirmed acid fast filamentous cultures. The part of the conventional Mycobacteriology of this work was published by EL Tigani-Asil et al. (2012, 2013. References strains M. farcinogenes (M39), previously typed by Hamid et al. (2002), was used as a positive control for typing BF agents following 16S-23S rDNA ITS method described by Roth et al. (1998). M. bovis (M116), characterized by Salih et al. (2005) was also used for comparison with the NTM PCR products.

Agarose Gel Preparation
Amount of 1.5g of agarose (Pharmacia Biotech AB , Uppsala , Sweden) was added to the mixture of 5ml of 10X TBE (0.023 M. Tris -Borate, 0.5 mM EDTA) buffer and 45 ml of distilled water, the mixture was melted in microwave oven for 2-5minutes, after cooling gel down to 60-70 ºC , 3 μl of ethidium bromide (0.5mg/ml) was added then the 50 ml of molten cooled agarose was loaded the 50ml gel cast (DNA plus, USA Scientific Int., Ocala, FL).

Electrophoresis and Gel Visualization
PCR products were electrophoresed in a 1.5% agarose gel and visualized using a UV source and compared to a molecular marker 100 bp. (Bioneer Co., Daejeon, South Korea).

Discussion
Until 15 years ago, species differentiation of mycobacteria was difficult especially for M. bovis. Identification of mycobacteria with the conventional phenotypic tools is time consuming and identification of closely related taxa is not always reproducible in contrast to molecular techniques (Roth et al., 2000). Nevertheless, conventional methods still have value in developing countries where modern laboratory tools are not available. PCR is a sensitive and specific tool in diagnosis of such fastidious organisms and reduces time for identification and perhaps the cost.